This research effort investigates how heme is distributed and utilized in the liver parenchymal cells. Experiments on intracellular heme distribution in vivo and with hepatocyte culture systems will concentrate on the transport of heme through the cytosol and across intracellular membranes, as well as across model membranes, i.e. of liposomes. Using a dual isotope pulse method in which 3H- and 14C- aminolevulinic acid serve as heme precursor, we have identified heme-binding proteins of the cytosol which may be implicated in intracellular heme transport. We are presently purifying and characterizing these potential heme carriers. We plan to use immunoprecipitation and the dual-label methodology to discover if the flow of endogenous and exogenous heme follows similar or divergent intracellular routes. Studies on heme utilization will concentrate on the turnover of the heme moiety of subforms of cytochrome P-450 which are major hepatic hemeproteins. In order to learn what determines the turnover of heme on these forms of cytochrome P-450, we will, by labeling the heme and the protein moiety, assess the catabolism of the protein moiety in relationship to that of heme. After investigating these aspects of intracellular heme transport and utilization under basal conditions, we will examine the same parameters under the influence of altered iron and porphyrin metabolism. Decreased hepatic heme levels will be produced by: 1) inhibiting heme synthesis, by limiting the iron supply, 2) blocking enzymatic steps of the synthesis with various porphyrogenic compounds, 3) increasing the cellular heme demand by inducing cytochrome P-450, and 4) decreasing microsomal heme levels by accelerating the catabolism of cytochrome P-450. The proposed work should ultimately furnish information about the physiology of intracellular heme transport and distribution, and should enhance our understanding of how aberrations of iron and porphyrin metabolism affect these processes. In addition, the research should provide important information for the rationale behind the use of heme infusions in the treatment of porphyria.